1. Field of the Invention
The inventive subject matter relates to a method of diagnosing Q fever and detecting exposure to Coxiella burnetii using specific immunodominant C. burnetti directly as antigen in antibody based assays such as enzyme-linked immunosorbent assays or western blot or the specific immunodominant C. burnetti proteins fused to an upstream biotinylated peptide.
2. Description of the Related Art
Coxiella burnetii is the causative agent of the disease Q fever and is found worldwide (1,2). The genus Coxiella is composed of the single species, C. burnetii, short, rod-shaped bacteria, 0.2 to 0.4 μm in diameter and 0.4 to 1.0 μm in length (1, 3). C. burnetii is typically maintained as an inapparent infection in domesticated animals, such as cattle, sheep and goats and is found in a number of tick species. The organism persists in feces, urine, milk and tissues so that fomites and infective aerosols are capable of easily forming. Disease occurs among farm workers in close proximity with the animals or their products. Transmission, therefore, is generally by inhalation of infected aerosols or even by the ingestion of raw milk that is infected with the bacteria. Additionally, the organism is highly infectious and is considered a potential biowarfare agent (4).
C. burnetii displays an antigenic phase variation, unlike other rickettsial species. In nature, the bacteria expresses phase I antigen, which is a polysaccharide component of the Coxiella lipopolysaccharide (5, 6, 7). After passage in culture, the bacteria changes from phase I to phase II. Phase II appears to be a manifestation of deletions of carbohydrate components from the lipopolysaccharide moiety (6).
Clinically, onset of Q fever occurs after an incubation of 18 to 21 days. Although rarely fatal, Q fever is typically abrupt, manifesting with fever, severe headache, chills, severe malaise, myalgia and chest pains. Acute and chronic infection, however, are characterized by different serological profiles (8). Early in the disease progression, Q fever resembles many infections such as influenza, salmonellosis, malaria, hepatitis, and brucellosis. For chronic Q fever, diagnosis is typically demonstrated by antibody against phase I and phase II antigens. In acute infection, diagnosis can typically only be made by direct detection of bacterial antigen or isolation of bacteria since significant serum antibodies are not available at this stage of infection.
Although it is advisable to diagnose Q fever early after infection, early after C. burnetii infection diagnosis is difficult due to the difficulty in bacterial isolation and antigen detection and because of the lack of seroconversion at these early stages. Specific complement fixation and agglutinating antibodies are useful in determining infection with agglutination test generally the more sensitive assay method (9-11). Additionally, the binding of fluorescent antibodies such as in indirect fluorescent assays (IFA) or enzyme-linked immunosorbent assays (ELISA) is also helpful in determining C. burnetii infection (12). Additional methods, such as direct tissue biopsy, culture of the organism and polymerase chain reaction (PCR) analysis are also useful but are used with much less frequency. The lack of popularity in using biopsy and culture is primarily due to the extensive equipment and expertise requirements as well as the time required to conduct the analysis. PCR offers high sensitivity and the ability to detect bacterial antigen early after infection. However, later in the course of the disease PCR's usefulness is considerably less likely to detect infection than serology (8, 13). Furthermore, PCR is often limited in use based on the availability of trained personnel and equipment.
Central to the development of improved detection and diagnostic immunoassay methods and standardization is the development of more effective antigens for use in existing antibody-based methods. Along these lines, efforts have been made to identify C. burnetii immunodominant proteins (14). Accordingly, a number of highly sera-reactive proteins have been identified, including chaperonin, translation elongation factor Tu and the 27 kDa outer membrane protein (Com-1) (14-16). Due to their high reactivity to anti-C. burnetii serum, these proteins have been suggested to be valuable antigens in detection and diagnostic assays. However, despite the utililty of immunoassays in the diagnosis of C. burnetii, currently available assays suffer from lack of sensitivity and reproducibility. Standardization of antigen will improve assay diagnostic performance and provide early and more accurate treatment regimens (13).
In order to improve standardization of diagnositic assays to C. burnetii and to increase assay sensitivity and specificity of detection, other C. burnetii immunodominant antigens need to be identified. Improved sensitivity can be achieved by defining antigens expressing a greater number of epitopes well represented in serum antibody repertoires. Alternatively or in addition, methods to ensure attachment of antigen to assay matrixes can be devised with improved orientation of the antigen on the assay matrix with concomitant reduction in steric hinderance in binding of specific anti-C. burnetii antibody in patient serum to the antigen.